Anti-inflammatory medicaments could be classified into those of steroid and of nonsteroidal type. Steroid anti-inflammatory compounds are still the most effective ones in the treatment of inflammatory diseases and conditions such as: asthma, chronic obstructive pulmonary disease, inflammatory nasal diseases such as allergic rhinitis, nasal polyps, intestinal diseases such as Crohn's disease, colitis, ulcerative colitis, dermatological inflammations such as eczema, psoriasis, allergic dermatitis, neurodermatitis, pruritus, conjunctivitis, autoimmune diseases such as rheumatoid arthritis, and inhibition of transplantation immunity. Moreover, steroids are used as adjunct chemotherapeutic agents in treating various malignancies, including leukemias, lymphomas, myelomas, and other malignancies of the hematopoietic system. In addition to excellent potency and effectiveness, medicaments of this type also possess numerous unfavourable side-effects, e.g., on carbohydrate metabolism, calcium resorption, secretion of endogenous corticosteroids as well as on the physiological functions of the pituitary gland, adrenal cortex and thymus. Recently developed steroids are highly effective against inflammatory conditions and processes since they inhibit many inflammation mediators, whereas their systemic side-effects are diminished. Patent applications WO 94/13690, WO 94/14834, WO 92/13873 and WO 92/13872 describe the so-called “soft” steroids or hydrolysable corticosteroids designed for topical application on the inflammation site, whereas their systemic side-effects are diminished due to instability of “soft” steroids in the serum, wherein the active steroid very rapidly hydrolyses into the inactive form. An ideal steroid, however, without unfavourable effects in a long-term and continuous treatment as required for the control of diseases such as asthma or Crohn's disease has yet to be found. Thus there is an acute need for steroids with an improved therapeutic profile, and/or fewer or milder side effects.
Nonsteroid anti-inflammatory medicaments having different mechanisms of action act on particular inflammation mediators, thus providing a therapeutic effect. Due to differences not only in mechanisms of action but also in the particular inflammation mediators inhibited, the steroid and nonsteroid medicaments possess different profiles of anti-inflammation effects, hence certain medicaments may be more suitable than others for particular conditions. Moreover, most nonsteroid anti-inflammatory medicaments are not absolutely specific and their use is accompanied by unfavourable side-effects when used in greater dosages or over long periods of time. It is known that many nonsteroid anti-inflammatory medicaments act as inhibitors of endogenous COX-1 enzyme, which is very important in maintaining the integrity of the gastric mucosa. Thus, the use of these medicaments often causes injuries of the gastric mucosa and even bleeding. ( Warner T. D. Proc. Natl. Acad. Sci. =l U.S.A. 1999, 96, 7563–7568.) Therefore, agents that selectively inhibit COX-2 but not COX-1 are preferable for treatment of inflammatory diseases Additionally, some anti-inflammatory compounds (such as theophylline) are known to have a very narrow therapeutic index, which limits their usage.
Recently, the nonsteroidal antiinflammatory drug celecoxib that specifically blocks COX-2 has been approved by the FDA for use in the treatment of rheumatoid arthritis (Luong et al. Ann. Pharmacother. 2000, 34, 743–760). COX-2 is also expressed in many cancers and precancerous lesions, and there is mounting evidence that selective COX-2 inhibitors may be useful for treating and preventing colorectal and other cancers (Taketo, M. M., J. Natl. Cancer Inst. 1998, 90, 1609–1620, Fournier et. al. J. Cell Biochem. Suppl. 2000, 34, 97–102).
In 1975, TNF-α was defined as an endotoxin-induced serum factor causing tumor necrosis in vitro and in vivo (Carswell E. A. et al. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3666–3670). In addition to antitumor activity, TNF-α has several other biologic activities, which are important in homeostasis as well as in pathophysiological conditions. The main sources of TNF-α are monocytes-macrophages, T-lymphocytes and mast cells.
The finding that anti-TNF-α antibodies (cA2) are effective in the treatment of patients suffering from rheumatoid arthritis (RA) (Elliot M. et al. Lancet 1994, 344, 1105–1110) intensified the interest to find new TNF-α inhibitors as possible potent medicaments for RA. Rheumatoid arthritis is an autoimmune chronic inflammatory disease characterized by irreversible pathological changes of the joints. In addition to RA, TNF-α antagonists are also applicable to several other pathological conditions and diseases such as spondylitis, osteoarthritis, gout and other arthritic conditions, sepsis, septic shock, toxic shock syndrome, atopic dermatitis, contact dermatitis, psoriasis, glomerulonephritis, lupus erhythematosus, scleroderma, asthma, cachexia, chronic obstructive lung disease, congestive heart failure, insulin resistance, lung fibrosis, multiple sclerosis, Crohn's disease, ulcerative colitis, viral infections and AIDS.
Two distinct retroviruses, human immunodeficiency virus (HIV) type-1 (HIV1) or type-2 (HIV-2), have been etiologically linked to the immunosuppressive disease, acquired immunodeficiency syndrome (AIDS). HIV seropositive individuals are initially asymptomatic but typically develop AIDS related complex (ARC) followed by AIDS. Affected individuals exhibit severe immunosuppression which predisposes them to debilitating and ultimately fatal opportunistic infections.
The disease AIDS is the consequence of HIV-1 or HIV-2 virus following its complex viral life cycle. The virion life cycle involves the virion attaching itself to the host human T-4 lymphocyte immune cell through the binding of a glycoprotein on the surface of the virion's protective coat with the CD4 glycoprotein on the lymphocyte cell. Once attached, the virion sheds its glycoprotein coat, penetrates into the membrane of the host cell, and uncoats its RNA. The virion enzyme, reverse transcriptase, directs the process of transcribing the RNA into single-stranded DNA. The viral RNA is degraded and a second DNA strand is created. The now double-stranded DNA is integrated into the human cell's genes and those genes are used for virus reproduction. RNA polymerase transcribes the integrated viral DNA into viral mRNA. The viral RNA is translated into the precursor gag-pol fusion polyprotein. The polyprotein is then cleaved by the HIV protease enzyme to yield the mature viral proteins. Thus, HIV protease is. responsible for regulating a cascade of cleavage events that lead to the virus particle's maturing into a virus that is capable of full infectivity.
The typical human immune system response, killing the invading virion, is taxed because the virus infects and kills the immune system's T cells. In addition, viral reverse transcriptase, the enzyme used in making a new. virion particle, is not very specific, and causes transcription mistakes that result in continually changed glycoproteins on the surface of the viral protective coat. This lack of specificity decreases the immune system's effectiveness because antibodies specifically produced against one glycoprotein may be useless against another, hence reducing the number of antibodies available to fight the virus. The virus continues to reproduce while the immune response system continues to weaken. In most cases, without therapeutic intervention, HIV causes the host's immune system to be debilitated, allowing opportunistic infections to set in. Without the administration of antiviral agents, immunomodulators, or both, death may result.
Hepatitis is an inflammation of the liver primarily caused by a virus and, less commonly, by certain medications or toxins (e.g. alcohol). The viral infection is often acquired through exposure to contaminated blood. Those most likely to contract the virus are intravenous drug users who share contaminated needles, although sexual contact with a person who has a form of Hepatitis can also spread the disease. In some instances, healthcare workers exposed to contaminated blood and persons who need repeated transfusions of blood have acquired a form of Hepatitis.
Three main types of viral Hepatitis have been identified, namely Hepatitis A, Hepatitis B and Hepatitis C, though at least four other viruses can cause hepatits. Hepatitis A is a highly infectious form of hepatitis and is the most common form of the disease. Hepatitis A is usually transmitted by contaminated food or water. The symptoms of hepatitis A often are similar to those of intestinal flu and a vast majority of persons with Hepatitis A recover completely.
Acute hepatitis B is potentially a more serious form of viral liver infection. Its symptoms are much the same as those of hepatitis A, but the symptoms are more severe and last longer. The primary initial symptoms of hepatitis A and hepatitis B include poor appetite, nausea, vomiting and fever. In later stages of hepatitis, the urine may become dark and persistent or recurring jaundice develops. In approximately 20% of cases of hepatitis cirrhosis (scarring of the liver) eventually develops. Cirrhosis as a result of hepatitis can be diagnosed through a blood test to evaluate liver function. Eventually, a liver affected by cirrhosis becomes tender as well. Hepatitis C, recognized as the major causative agent of non-A and non-B hepatitis, share common symptoms with hepatitis A and B. and patients develop chronic infections which can ultimately lead to liver cirrhosis.
Neoplastic diseases are a common cause of death, caused by autonomous, non-controlled division of cells. This division can be triggered by:                1. gene mutations caused by carcinogens        2. viruses        3. external signals which activate mitosis of certain cell type        
Neoplastic diseases are treated with various inhibitors of mitosis and cellular metabolism. However, specificity has been the major problem with anticancer agents. In the case of anticancer agents, the drug needs to distinguish between host cells that are cancerous and host cells that are not cancerous. The vast bulk of anticancer drugs are indiscriminate at this level. Typically anticancer agents have negative hematological effects (e.g., cessation of mitosis and disintegration of formed elements in marrow and lymphoid tissues), and immunosuppressive action (e.g., depressed cell counts), as well as a severe impact on epithelial tissues (e.g., intestinal mucosa), reproductive tissues (e.g., impairment of spermatogenesis), and the nervous system. P. Calabresi and B. A. Chabner, In: Goodman and Gilman The Pharmacological Basis of Therapeutics (Pergamon Press, 8th Edition) (pp. 1209–1216). Success with chemotherapeutics as anticancer agents has also been hampered by the phenomenon of multiple drug resistance, resistance to a wide range of structurally unrelated cytotoxic anticancer compounds. J. H. Gerlach et al., Cancer Surveys, 5:25–46 (1986). The underlying cause of progressive drug resistance may be due to a small population of drug-resistant cells within the tumor (e.g., mutant cells) at the time of diagnosis. J. H. Goldie and Andrew J. Coldman, Cancer Research, 44:3643–3653 (1984). Treating such a tumor with a single drug first results in a remission, where the tumor shrinks in size as a result of the killing of the predominant drug-sensitive cells. With the drug-sensitive cells gone, the remaining drug-resistant cells continue to multiply and eventually dominate the cell population of the tumor. Finally, the treatment of cancer has been hampered by the fact that there is considerable heterogeneity even within one type of cancer. Some cancers, for example, have the ability to invade tissues and display an aggressive course of growth characterized by metastases. These tumors generally are associated with a poor outcome for the patient. And yet, without a means of identifying such tumors and distinguishing such tumors from non-invasive cancer, the physician is at a loss to change and/or optimize therapy. What is needed is a specific anticancer approach that is reliable for a wide variety of tumor types, and particularly suitable for invasive tumors. Importantly, the treatment must be effective with minimal host toxicity. Thus it is necessary to overcame the potential of cells to decrease the intracellular amount of the active drug, to improve cellular targeting and/or to improve pharmacokinetic of antineoplastic drug.
Macrolides such as macrolide antibiotics accumulate within different cells of subjects administered such molecules, especially within phagocyte cells such as mononuclear peripheral blood cells, polymorphonuclear cells, peritoneal and alveolar macrophages as well as in the liquid surrounding the bronchoalveolar epithelium (Glaude R. P. et al., Antimicrob. Agents Chemother., 33 1989, 277–282; Olsen K. M. et al., Antimicrob. Agents Chemother., 40 1996, 2582–2585). Moreover, relatively weak inflammatory effects of some macrolides have been described. For example, the anti-inflammatory effect of erythromycin derivatives (J. Antimicrob. Chemother., 41 1998 37–46; WO 00/42055) and azithromycin derivatives has recently been described (EP 0283055). Anti-inflammatory effects of some macrolides are also known from in vitro and in vivo studies in experimental animal models such as zimosane-induced peritonitis in mice (J. Antimicrob. Chemother., 30 1992 339–348) and endotoxin-induced neutrophil accumulation in rat trachea (J. Immunol. 159 1997 3395–4005). The modulating effect of macrolides upon cytokines such as interleukin 8 (IL-8) (Am. J. Respir. Crit. Care Med. 156 1997 266–271) or interleukin 5 (IL-5) (EP 0775489 and EP 0771564) is known as well. Additionally, the favorable pharmacokinetic profile of macrolides could elevate tissue concentration of a compound e.g. in the liver, or increase white blood cells/plasma ratio (Girard A. E. et al., Antimicrob. Agents Chemother. 31 1987 1948–54; Widlfeuer A. et al, Antimicrob. Agents Chemother. 40 1996, 75–79).
In order to obtain compounds with improved/novel activity profile towards diseases where selective activity is needed several different active substances have been connected to macrolides with different types of linkers. A few examples of hybrids/conjugates/chimeras of erythromycin A derivatives and nucleobases (uracil and thymine) or thymidine-derived nucleosides have been reported. (Costa A. M. at al. Tetrahedron Lett. 41, 2000, 3371–3375). However, such constructs did not show activity/selectivity toward a desired target. Moreover, macrolide constructs where the linker would be of the peptide type not been reported.
The peptide linker introduced in our hybrid molecules as the linker enables them to act as prodrugs, releasing the V moiety by specific lysosomal cleavage within the target cell. Similar linkers have been described for other small molecules (in our case represented by hybrids of an anti-inflammatory, antineoplastic and antiviral compound) and a macromolecule or polymer (Duncan R. et al. in Robinson J. R. and Lee V. H. (eds.) Controlled Drug Delivery:Fundamentals and Applications, 2nd edition, 1987 581–607, Subr V. et al, J. Controlled Rel. 18 1992 123–132)